Figure 5. Granulocyte-monocyte potentials segregate prior to dissociation from lymphoid and erythro-megakaryocytic lineages.

(a) GSEA-based comparison of GE^- and GE⁺ pre-GM gene expression using CLP (top) and pre-Meg-E (bottom) gene sets. Normalized enrichment score (NES) and P-value are indicated. (b-e) Colony formation evaluated after 8 days of culture of indicated progenitor cells. Type of colonies assayed are (b) myeloid (CFU-GM), (c) erythroid (BFU-E), (d) megakaryocyte (CFU-Mk) and (e) combined myeloid and erythroid (CFU-Mix). (f) Cytospins from single CFU-Mix colonies derived from pre-GM GE⁺, showing combined erythroid cells (Ery), polymorphonuclear cells (PMN), megakaryocytes (Meg) and mast cells (Ma) but no monocytes. Scale bars: 25μm. (g) Assessment of lymphoid potential of the indicated progenitors. For each population the indicated number of cells/well were plated under B-cell (OP9 stroma) and T-cell (OP9-DL1 stroma) conditions. Bars indicate the percentage of wells producing B-cells (defined as CD19⁺) (top) or T-cells (defined as Thy1.2⁺CD25⁺ or CD4⁺CD8⁺) (bottom), respectively. Mean (±s.e.m.) number of positive wells plated with 1, 10 or 50 cells. Data are from 2 experiments each with 24 wells/population and cell density. Number of CFU-GM, BFU-E and CFU-Mk colonies are shown as mean ±s.e.m of 6,7,6,8,4 (b), 8,7,6,6,4 (c) and 7,7,7,6,4 (d) biological replicates for pre-GM GE^-, pre-GM GE⁺, GMP GE^-, GMP GE⁺ and pre-Meg-E resp. from 4 experiments. Numbers of CFU-mix colonies are mean ±SD from 3 biological replicates, each assayed in technical duplicate.